Jean-Claude Louis

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

BOTH FAMILIAL PARKINSON'S DISEASE MUTATIONS ACCELERATE ALPHA -SYNUCLEIN AGGREGATION

Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant α-synuclein form insoluble fibrillar aggregates with antiparallel β-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of α-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease.

Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD. α-Synuclein fibrils similar to the Lewy body filaments can be formedin vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of α-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated α-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to α-synuclein concentration, and (iii) mutant α-synuclein can seed the aggregation of wild type α-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with α-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of α-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of α-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, α-synuclein nucleation may be the rate-limiting step for the formation of Lewy body α-synuclein fibrils in Parkinson’s disease.

Parkinson's Disease-associated α-Synuclein Is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs

Parkinsons disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in α-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes β- and γ-synuclein. α-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for β- or γ-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between α- and β-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of α-, β-, and γ- synuclein show the same natively unfolded structure. While over time α-synuclein forms the previously described fibrils, no fibrils could be detected for β- and γ-synuclein under the same conditions. Most importantly, β- and γ-synuclein could not be cross-seeded with α-synuclein fibrils. However, under conditions that drastically accelerate aggregation, γ-synuclein can form fibrils with a lag phase roughly three times longer than α-synuclein. These results indicate that β- and γ-synuclein are intrinsically less fibrillogenic than α-synuclein and cannot form mixed fibrils with α-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to α-synuclein and are also abundant in brain.

Inhibition of Phosphatidylinositol 3-Kinase Activity Blocks Cellular Differentiation Mediated by Glial Cell Line-Derived Neurotrophic Factor in Dopaminergic Neurons

Abstract: Glial cell line‐derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3‐kinase activity in GDNF‐stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3‐kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µM 2‐(4‐morpholinyl)‐8‐phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3‐kinase activity, completely inhibited GDNF‐induced phosphatidylinositol 3‐kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase‐immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF‐induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase‐immunoreactive neurons. Our findings show that GDNF‐induced differentiation of dopaminergic neurons requires phosphatidylinositol 3‐kinase activation.

Inhibition of Glial Cell Line‐Derived Neurotrophic Factor Induced Intracellular Activity by K‐252b on Dopaminergic Neurons

Abstract: The c‐ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line‐derived neurotrophic factor (GDNF). K‐252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain‐derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin‐3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K‐252b would modulate GDNF‐induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 µM K‐252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K‐252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase‐immunoreactive neurons. K‐252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K‐252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K‐252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.

Distribution of immunophilin FKBP-12 protein and mRNA within the mammalian cochlea and cochlear nucleus

Immunophilin FK binding protein-12 (FKBP-12), the soluble receptor for the immunosuppressant drug FK506, is involved in a number of neuronal activities including increased nerve regeneration in the peripheral nervous system and enhanced recovery in animal models of neurodegenerative diseases. In addition, FKBP-12 is tightly bound to the calcium release channel ryanodine receptor and physiologically interacts with the inositol 1,4,5-trisphosphate receptor. In nearly all cell types, release of intracellular Ca(2+) and subsequent second messenger signaling involves activation of these ion channels. We determined the distribution of FKBP-12 within the mammalian cochlea and dorsal cochlear nucleus (DCN) in order to gain insight into Ca(2+) regulation within the cochlea and to possibly identify potential cellular targets for neuroimmunophilin ligands that may prove useful in protection and recovery following ototoxic insult. FKBP-12 protein and mRNA were found to be abundant throughout rat and guinea pig cochlea and DCN.

α-Synuclein fibrillogenesis as target for drug development

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most frequent neurodegenerative disorders. Current symptomatic treatment of PD is useful for a limited period during disease progression, and the state-of-the-art symptomatic treatment of AD with cholinesterase inhibitors is only marginally effective. Thus, a major unmet medical need exists for both diseases and the ideal therapeutic would halt disease progression and not just alleviate symptoms. Development of a disease-modifying treatment requires some understanding of the disease etiology. For AD the amyloid cascade hypothesis [1] can serve as a framework from which targets for therapeutic intervention can be developed. In contrast, there has been no unifying hypothesis for the pathogenesis of PD, and therapeutic targets still have to be defined. Here we describe basic mechanistic studies which lead us to propose α-synuclein fibrillogenesis as a target for therapeutic intervention for PD and other α-synucleinopathies, e.g., dementia with Lewy bodies.